A defined amino acid exchange close to he putative nucleotide binding site is responsible for an oxygen-tolerant variant of the Rhizobium meliloti NifA protein

Abstract

In Rhizobium meliloti the NifA protein plays a central role in the expression of genes involved in nitrogen fixation. The R. meliloti NifA protein has been found to be oxygen sensitive and therefore acts as a transcriptional activator only under microaerobic conditions. In order to generate oxygen-tolerant variants of the NifA protein a plasmid carrying the R. meliloti nifA gene was mutagenized in vitro with hydroxylamine. About 70 mutated nifA genes were isolated which mediated up to 12-fold increased NifA activity at high oxygen concentrations. A cloning procedure involving the combination of DNA fragments from mutated and wild-type nifA genes allowed mapping of the mutation sites within the central part of the nifA gene. For 17 mutated nifA genes the exact mutation sites were determined by DNA sequence analysis. It was found that all 17 mutated nifA genes carried identical guanosine--adenosine mutations resulting in a methionine--isoleucine exchange (M217I) near the putative nucleotide binding site within the central domain. Secondary structure predictions indicated that the conformation of the putative nucleotide binding site may be altered in the oxygen-tolerant NifA proteins. A model is proposed which assumes that at high oxygen concentrations the loss of activity of the R. meliloti NifA protein is due to a conformational change in the nucleotide binding site that may abolish binding or hydrolysis of the nucleotide. Such a conformational change may be blocked in the oxygen-tolerant NifA protein, thus allowing interaction with the nucleotide at high oxygen concentrations.

Genetic regulation of nitrogen fixation in rhizobia.

“…Fig. 6) (215). As an attractive model it has been proposed that this mutation locks the nucleotide-binding site in a conformation that allows binding or hydrolysis of ATP even under aerobic conditions whereas under the same conditions this interaction is prevented in the wild-type protein by a conformational change induced by oxidation of the postulated redox-sensing metal ion.…”

Role of the transcriptional activator RocR in the arginine‐degradation pathway of Bacillus subtilis

“…2). None of the mutations correspond to residues identified by constitutive mutations in regulatory proteins such as XylR (Delgado et al, 1995;Fernandez et al, 1995), NtrC (Dixon et al, 1991;Flashner et al, 1995;Klose et al, 1993;Moore et al, 1993;Weglenski et al, 1989), DmpR (Shingler and Pavel, 1995), or NifA (Krey et al, 1992). The ␤-galactosidase activity was measured in strain QB5570 transformed, separately, with each of the following plasmids: pROC1 (i.e.…”

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

“…Moreover, Oliveira et al (18) produced mutants for each of the four cysteine residues of the putative Fe-S cluster binding motif in H. seropedicae NifA, and observed that the mutant proteins were still able to bind DNA although the ability to activate transcription was completely abolished. Oxidation of the Fe-S cluster may trigger conformational changes affecting nucleotide binding and/or hydrolysis necessary for transcriptional activation, as proposed (18,20). Therefore, under aerobic conditions, the oxidized Fe-S cluster would lead to a transcriptionally inactive form of NifA possibly compromising the catalytic activity of the central domain.…”